The fact that in diabetic patients an increase in the glycohemoglobin content of the erythrocytes takes place affords a method of determination useful in the control of the metabolism of diabetic patients. Through a nonenzymatic, irreversible reaction of glucose in the blood with one of the terminal amino groups of the protein chains and with the .epsilon.-amino group of lysine, glycosylated hemoglobin fractions are formed in the erythrocytes. These fractions may be separated by means of chromatography from normal hemoglobin. The glycoprotein synthesis takes place over the entire half-life period of 120 days of the erythrocytes, with the reaction being a function of the average glucose concentration and its duration in the blood. The total HbA.sub.1 glycohemoglobin fraction consists of three subfractions, HbA.sub.1a +HbA.sub.1b +HbA.sub.1c. The glycosylated hemoglobin designated by HbA.sub.1c is particularly well-suited for the control of blood sugar. The HbA.sub.1c content, which is present in an amount of 3-6% in the total hemoglobin of normal adults, is therefore an indication of the average level of blood sugar over the life of the erythrocytes. The determination of the value of HbA.sub.1c is effected after hemolysis of the erythrocytes, such as by means of chromatographic separation of the hemoglobin fractions in macrocolumns [Trivelli et al., New Engl. J. Med. 284, 353-357 (1971)] or in microcolumns and subsequent colorimetric measurements in a spectrophotometer at approximately 415 nm.
Separation by means of microcolumns has largely superseded the macrocolumn method, which is expensive in practice, since it has been determined that the total fraction of the glycosylated hemoglobin HbA.sub.1a+1b+1c also affords the long-term or chronic blood sugar value in a good correlation; accordingly, the separation of the subfractions, which is not possible in microcolumns, may be eliminated for diagnostic purposes [Koening, R. J., C. M. Peterson, R. L. Jones, C. Saudek, M. Lehrman and A. Cerami, "Correlation of glucose regulation and hemoglobin A.sub.1c in diabetes mellitus", N. Eng. J. Med. 295, 417-420 (1976); Fitzgibbons, J. F., F. D. Koler and R. T. Jones, "Red cell age-related changes of hemoglobins A.sub.1a+b and A.sub.1c in normal and diabetic subjects", J. Clin. Inves. 58, 820-824 (1976)].
All of the commerically available microcolumn determination sets of the various manufacturers use the same fundamental technique. The HbA.sub.1 total fraction is separated from the residual hemoglobin by means of an ion exchanger, with the aid of a buffer with its pK value adjusted to the ionic strength of the exchanger. As the reference value, the residual hemoglobin is then washed from the column with a second elution solution, or the total hemoglobin is determined from a corresponding hemolysate aliquot. The relative proportion of the HbA.sub.1 fraction is then calculated for the total hemoglobin from both values. Fundamentally, methods of determination wherein the hemoglobin bonded to the exchange resin is removed by centrifugation are also based on this technique (for example, Leeco Diagnostics).
It is known that the glycosylation of hemoglobin in the erythrocytes takes place by a two-stage process, wherein among others, initially the aldehyde group of the glucose reacts with the amino group of the terminal valine of the .beta.-chain of the hemoglobin to give the aldimine form (Schiff base). This relatively unstable aldimine derivative is then rearranged by means of an Amadori transposition into the relatively stable ketoamine form. Short term blood sugar fluctuations during the day on which the test is performed may thus lead to a correspondingly high rate of the formation of the unstable aldimine, which could render the evaluation of the HbA.sub.1 value as a long-term parameter of diabetic metabolism control questionable. See Schernthaner, Dtsch. Med. Wschr. 106, 259-261 (1981); J. Ditzel, Diabetologia 19, 403-404 (1980).